Method for predicting early-stage hypertensive kidney damage based on urinary peptide determination and application

Abstract

The invention relates to the technical field of renal injury prediction, in particular to a method for predicting early hypertension renal injury based on urinary peptide determination and application. The method comprises the following steps: S1, collecting and detecting urine and blood of hypertension patients, respectively collecting data of normal people, hypertension people and hypertension and kidney disease groups, and screening; s2, the health condition of the hypertensive patient is confirmed through follow-up visit; s3, preparing a urine sample and testing. According to the method for predicting the early-stage hypertensive kidney damage based on the urine peptide determination and the application, the urine peptide determination is applied to the hypertensive patient, so that the kidney damage of the hypertensive patient can be predicted in an earlier stage.

Description

Technical Field

[0001] The present invention relates to the technical field of kidney damage prediction, and in particular to a method and application of predicting early hypertensive kidney damage based on urine peptide determination. Background Art

[0002] Hypertension (HTN), especially essential hypertension, is a common chronic cardiovascular disease in modern society, affecting the health of hundreds of millions of people worldwide. Hypertension refers to a pathological state in which systolic blood pressure is ≥140 mmHg and / or diastolic blood pressure is ≥90 mmHg without the use of antihypertensive drugs. Long-term hypertension increases the risk of complications such as heart disease, stroke and kidney disease.

[0003] The kidney plays a vital role in the pathophysiology of hypertension. The kidney is not only a key organ for regulating sodium-water balance in the body, but also affects blood pressure by regulating the renin-angiotensin-aldosterone system (RAAS). Long-term hypertension leads to chronic changes in kidney structure and function, which may eventually lead to kidney damage.

[0004] In recent years, renal damage has been recognized as one of the common complications of essential hypertension. Renal damage not only exacerbates the severity of hypertension but also increases the risk of cardiovascular events.

[0005] However, the onset of hypertensive nephropathy is often hidden, and most patients are not discovered until they develop renal insufficiency, which may lead to multiple complications and a high mortality rate. Therefore, early diagnosis and intervention are crucial to improving the prognosis of patients with hypertensive nephropathy. Serum creatinine (Scr) levels are often used as an indicator of renal damage, but their low sensitivity and specificity hinder the accurate assessment and timely reflection of renal function and structure, which can easily lead to missed diagnosis and delayed disease treatment;

[0006] Studies have shown that CKD273 in urine has universal and routine applicability in evaluating renal damage in CKD patients, and can accurately identify and evaluate immune-related CKD, such as IgA nephropathy, membranous nephropathy, focal segmental sclerotic glomerulonephritis, and lupus nephritis. CKD237 in urine is an early and reliable biomarker for the risk of kidney disease. In addition, it is also helpful in evaluating the effectiveness of treatment and predicting the prognosis of non-immune kidney diseases (such as diabetic nephropathy).

[0007] Huang et al. found that CKD273 has multiple functions and can not only predict the prognosis of kidney disease, but also plays a vital role in diagnosing kidney damage in heart transplant patients. In addition, it can also be used to predict the prognosis of cardiovascular disease in patients with kidney disease. CKD273 is highly significant in guiding the use of renin-angiotensin-aldosterone system inhibitors, dapagliflozin and other drugs in patients with diabetes and renal hypertension. The study of CKD273 confirmed that urinary peptides have significant value in predicting the prognosis of cardiovascular disease and certain kidney diseases. Based on this information, it is hypothesized that there may be a relationship between urinary peptides and hypertension-related kidney damage.

[0008] To this end, a method and application based on urine peptide determination for predicting early hypertensive renal damage was designed to provide another technical solution to the above technical problems. Summary of the invention

[0009] Based on this, it is necessary to provide a method and application based on urine peptide determination for predicting early hypertensive renal damage in response to the above technical problems, so as to solve the technical problems raised by the above background technology.

[0010] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0011] A method for predicting early hypertensive renal damage based on urine peptide measurement, the steps are as follows:

[0012] S1: Collect test data of patients with hypertension;

[0013] S2: Follow up hypertensive patients and confirm their kidney damage;

[0014] S3: Prepare urine sample and perform test.

[0015] As a preferred embodiment of the method for predicting early hypertensive renal damage based on urine peptide determination provided by the present invention, in the step S1, data of hypertensive patients are collected and tested, and the steps are as follows:

[0016] Urine and blood of hypertensive patients were collected and tested, and data of normal population, hypertensive population and hypertension with kidney disease group were collected and screened to form test group data and control group data.

[0017] As a preferred embodiment of the method for predicting early hypertensive renal damage based on urine peptide determination provided by the present invention, the data of the test group and the control group respectively include the patient's name, gender, age, HTN duration and hypertension grade, hypertensive fundus lesions, urine microalbumin / creatinine, serum β2-MG, Scr, blood urea nitrogen, homocysteine ​​and cystatin C levels, and the estimated glomerular filtration rate calculated using CKD-EPI.

[0018] As a preferred embodiment of the method for predicting early hypertensive renal damage based on urine peptide determination provided by the present invention, hypertensive patients with normal albuminuria are followed up every 6 months, and blood and first morning urine samples are collected at the same time, and the samples are centrifuged, packaged and frozen for storage.

[0019] As a preferred embodiment of the method for predicting early hypertensive renal damage based on urine peptide determination provided by the present invention, a urine sample is prepared and tested, and the steps are as follows:

[0020] A) Bond Elut C18 solid phase extraction cartridges were used to pretreat thawed urine samples at room temperature;

[0021] B) Using micro-liquid chromatography, 10 μL of urine sample is injected into the mass spectrometer

[0022] C) quantitative analysis of peak area;

[0023] D) The abundance values ​​of identified peptides in each sample were combined and quantified using urine creatinine levels;

[0024] E) Comparisons between different groups.

[0025] An application based on urine peptide determination in predicting early hypertensive renal damage, applying urine peptide determination in kidney testing.

[0026] It can be seen without a doubt that the above-mentioned technical solution of the present application can definitely solve the technical problem to be solved by the present application.

[0027] At the same time, through the above technical solutions, the present invention has at least the following beneficial effects:

[0028] The present invention provides a method and application for predicting early hypertensive renal damage based on urine peptide determination. By applying urine peptide determination in hypertension, a more forward-looking prediction of the kidneys of hypertensive patients can be made. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0030] Figure 1 is a flow chart of the present invention;

[0031] Figure 2 It is a schematic diagram of comparison of baseline data of participants of the present invention;

[0032] Figure 3 A comparative diagram of urine peptide levels of 319 participants of the present invention;

[0033] Figure 4 A schematic diagram of the demographic characteristics of patients with different urine peptide levels of the present invention;

[0034] Figure 5 This is a schematic diagram of the risk of new-onset proteinuria in HTN+ hypertensive patients of the present invention;

[0035] Figure 6 This is a schematic diagram of the correlation analysis between urine peptides and urine microalbumin / creatinine in patients with hypertension according to the present invention;

[0036] Figure 7 To compare the sensitivity and specificity of different indicators of the present invention in diagnosing hypertensive renal damage;

[0037] Figure 8 This is a schematic diagram of the correlation analysis between urine peptides of hypertensive patients and estimated glomerular filtration rate of the present invention;

[0038] Fig. 9 The ROC curve diagram of different indicators for diagnosing early hypertensive renal damage in the present invention;

[0039] Fig.10 This is a schematic diagram of the first experimental data of the present invention, in which 4 sequences in Supplementary table 1-5 correspond to 23 polypeptides, and 23 polypeptides correspond to 8 proteins;

[0040] Fig.11 This is a schematic diagram of the second experimental data of the present invention, in which 4 sequences in Supplementary table 1-5 correspond to 23 polypeptides, and 23 polypeptides correspond to 8 proteins;

[0041] Fig.12 This is a schematic diagram of the third experimental data of the present invention, in which 4 sequences in Supplementary table 1-5 correspond to 23 polypeptides, and 23 polypeptides correspond to 8 proteins;

[0042] Fig.13 This is a schematic diagram of representative results of 5 cases in each group in Supplementary table-2 of the present invention. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0044] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings.

[0045] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features and technical solutions in the embodiments may be combined with each other.

[0046] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0047] Embodiment 1

[0048] Reference Figure 1-Figure 13 , a method based on urine peptide measurement in predicting early hypertensive renal damage.

[0049] Collect and test urine and blood from patients with hypertension, collect data from normal people, hypertensive people, and hypertension with kidney disease, and screen them to form test group data and control group data;

[0050] Data for the test group were collected from treated hypertensive patients and age-matched healthy individuals who underwent physical examinations at the Third Affiliated Hospital of Chongqing Medical University and Jiulongpo District People's Hospital (Chongqing, China) from October 2016 to October 2019. The study was approved by the local ethics committee (approval number: 2020KLS15); the inclusion criteria for hypertensive patients were: meeting the diagnostic criteria and classification of the 2013 ESH / ESC hypertension guidelines, for example, office SBP ≥ 140 mmHg and / or office DBP ≥ 90 mmHg; 24-hour ambulatory SBP ≥ 130 mmHg and / or 24-hour ambulatory DBP ≥ 80 mmHg; and home SBP ≥ 135 mmHg and / or home DBP ≥ 85 mmHg; hypertensive patients aged 60-75 years, regardless of gender, with a history of hypertension for at least 10 years.

[0051] The data in the test group are filtered as follows:

[0052] Exclusion criteria included secondary hypertension, such as renal hypertension, secretory hypertension, vascular hypertension, and hypertension caused by drugs and sleep apnea, diabetes, primary glomerular disease and other secondary glomerular diseases, chronic wasting diseases (such as malignant tumors and tuberculosis), autoimmune diseases, severe heart disease, and liver failure.

[0053] The data collection and screening of the control group are as follows:

[0054] People who received a comprehensive physical examination at the hospital's physical examination center were excluded from the examination after hypertension, diabetes, liver, heart, various kidney diseases (including acute and chronic infections secondary to surgery), tumors, and other diseases. After collecting urine for testing, their urine peptide levels were compared with those of other groups.

[0055] The included patients with hypertensive nephropathy were excluded from renal damage caused by other reasons, based on the history of hypertension, family history of hypertension and other target organ damage of hypertension. Among them, 32 cases were diagnosed with hypertensive nephropathy after excluding other glomerular diseases by renal biopsy. Renal biopsy showed enlarged glomerular volume, ischemic shrinkage of the basement membrane of some glomerular capillaries, thickening of arteriolar walls, proliferation of mesangial cells, increase of mesangial matrix, and hyaline degeneration of renal arterioles.

[0056] Data collected for the test and control groups included patient name, sex, age, HTN duration and hypertension grade, hypertensive fundus lesions, urine microalbumin / creatinine (uACR), serum β2-MG, Scr, blood urea nitrogen (BUN), homocysteine ​​(Hcy) and cystatin C levels, and estimated glomerular filtration rate (eGFR) calculated using the CKD Epidemiology Collaboration (CKD-EPI). Proteinuria was divided into normal proteinuria (uACR < 30 mg / g) and low proteinuria (UACR 30-300 mg / g), and when uACR > 300 mg / g, it was defined as high albuminuria.

[0057] In this embodiment, the diagnostic criteria for hypertensive renal damage are as follows: renal biopsy shows changes in hypertensive renal damage, and other glomerular diseases are excluded by renal biopsy; renal biopsy shows enlarged glomerular volume, ischemic shrinkage of glomerular capillary basement membrane, thickening and hyalinization of arteriolar walls, proliferation of mesangial cells and matrix, or the patient has no renal biopsy but has a history of hypertension (SBP and DBP>140 / 90mmHg) for at least 10 years, a family history of hypertension, mild to moderate proteinuria 10 years after hypertension, or left ventricular hypertrophy, retinal arteriosclerosis, and atherosclerosis caused by hypertension.

[0058] Follow-up of patients

[0059] All hypertensive patients with normoalbuminuria were followed up every 6 months, and their blood and first morning urine samples were collected, which were centrifuged, aliquoted and cryopreserved. Urine polypeptides, uACR, serum B2-MG, Scr, BUN, Hcy and CysC in blood samples were tested. Patients who were first found to have uACR ≥ 30 mg / g underwent urine uACR testing for three consecutive months. The primary endpoint was uACR ≥ 30 mg / g, and the secondary endpoint was a decrease in eGFR ≥ 30%.

[0060] Detection of urine peptides

[0061] SKYLINE software was used to predict potential peptide fragments. Urine samples were subjected to liquid chromatography-tandem mass spectrometry (LC / MS), and data were acquired using a triple quadrupole 6600+ mass spectrometer (Sciex 2.0.1, Framingham, MA, USA).

[0062] The urine sample preparation and testing procedures are outlined below:

[0063] Step 1: Bond Elut C18 solid phase extraction column (3mL / 200mg, Agilent, USA) Urine samples were thawed to room temperature and pretreated by solid phase extraction; 1mL of methanol and 1mL of ultrapure water were added to the solid phase extraction column in sequence; 200μL of urine was taken, 800μL of ultrapure water was added, and the mixture was mixed by oscillation for 1min before loading and dripping under gravity; 1mL of ultrapure water was used for elution, the mixture was dripped under gravity and then vacuum-evacuated; elution: 1.5mL of 70% methanol was used for elution and the eluate was collected; the mixture was concentrated by rotary vacuum centrifugation for 4h to dryness; 50μL of 50% methanol was added, the mixture was oscillated for 1min, ultrasonicated for 2min, centrifuged at 13000×g for 10min, and 2μL of the supernatant was taken for mass spectrometry analysis.

[0064] Step 2: IDA mass spectrometry method: Use the IDA method to collect samples on the SCIEX TripleTOFTM 6600+ and adjust the parameters.

[0065] Step 3: Use SCIEXOS2.0.1 software to quantitatively analyze the peak area.

[0066] The abundance values ​​(peak areas) of the identified peptides in each sample were then combined and numerically normalized using urine creatinine levels (to minimize any bias caused by variations in urine volume among patients)

[0067] Finally, comparisons between the different groups were performed.

[0068] result

[0069] 319 participants, including 54 healthy people in the normal control group, 167 patients in the non-albuminuria hypertension group, 51 patients in the hypertension + microalbuminuria group, and 47 patients in the hypertension + macroalbuminuria group. Urinary peptides were detected in all participants. Among the 167 non-albuminuria hypertensive patients, 20 patients were dropped out due to loss to follow-up or withdrawal from the study. A total of 147 hypertensive patients were followed up for 2.5-5.5 years ( Figure 1 ), there were no significant differences in age, gender and body mass index between healthy people and hypertensive patients. Among hypertensive patients, the proportion of hypertensive retinopathy in the hypertension + microalbuminuria group and the hypertension + macroalbuminuria group was significantly higher than that in the hypertension group with normal albuminuria; the levels of Hcy, CysC, B2-MG and uACR in patients with hypertension and proteinuria were significantly higher than those in the hypertension group with normal albuminuria. Figure 2 The table in shows the baseline data comparison.

[0070] refer to Figure 1 Of the 167 HTN patients, 20 were lost during follow-up or dropped out of the study, and 2 died of unknown causes, which were not related to renal dysfunction. The 147 patients with normoproteinuria HTN were followed up for an average of 3.8 years, of which 35 patients showed uACR ≥ 30 mg / g for three consecutive times. The ROC curve was used to determine the critical value based on sensitivity and specificity.

[0071] The prediction threshold of urine polypeptide for new-onset proteinuria in HTN patients is 0.097 e+06 , patients were divided into high-risk (urinary polypeptide ≥ 0.097 e+06 ) group and low risk (urinary polypeptide <0.097 e+06 ) group. In the normoalbuminuria hypertension group, there were 39 high-risk patients and 108 low-risk patients.

[0072] Age, sex, BMI, smoking history, follow-up time, type of antihypertensive medication, BUN level, Scr level, eGFR level, and the proportion of eGFR reduction ≥ 30% were not significantly different between high-risk and low-risk patients. However, compared with low-risk patients, high-risk patients had higher rates of hypertensive retinopathy, Hcy, CysC, β2-MG, and uACR ≥ 30 mg / g (P ﹤ 0.05). In the high-risk (n = 39) and low-risk (n = 108) groups, ACR ≥ 30 mg / g accounted for 76.9% (30 / 39) of the high-risk group and 4.6% (5 / 108) of the low-risk group ( Figure 3 ).

[0073] Cox regression model was used to analyze the occurrence of new-onset proteinuria in patients with different risks, adjusting for age, sex, duration of hypertension, proportion of hypertensive retinopathy, and eGFR. In patients with normoalbuminuria and hypertension, the incidence of new-onset proteinuria was significantly higher in the high-risk group than in the low-risk group (P = 0.001; Figure 5 ). The relationship between urine polypeptides and ACR and eGFR in patients with hypertension was further analyzed. It was found that the urine polypeptide level was positively correlated with ACR (r = 0.494, P = 0.01), but had no significant correlation with eGFR (r = -0.102, P = 0.217; ref. Figure 6 and Figure 8 . )

[0074] Embodiment 2

[0075] An application of urine peptide determination in predicting early hypertensive renal damage.

[0076] ROC curve analysis for the diagnosis of early hypertensive renal damage showed that the AUC value of urine peptide was 0.925 (the cutoff value was 0.097 e+06 , sensitivity = 0.80, specificity = 0.96), the AUC for Hcy was 0.753, (cutoff value was 15.5 μmol / L, sensitivity = 0.84, specificity = 0.52), for B2-MG, the AUC was 0.796 (cutoff value = 4.8 mg / L, sensitivity = 0.51, specificity = 0.90), and for Cysc, the AUC was 0.769 (cutoff value = 1.21 mg / L, sensitivity = 0.60, specificity = 0.90); Fig. 9 and Figure 7 in the table).

[0077] Hypertension is a risk factor for cardiovascular morbidity and mortality. The kidney is not only an important organ involved in the pathophysiological changes of hypertension, but also an important target organ of hypertension. However, patients with early hypertensive renal damage have atypical clinical symptoms and fewer changes in renal structure and morphology. Therefore, it is crucial to identify biomarkers or indicators for early detection of renal damage.

[0078] The present invention includes hypertensive patients and age-matched healthy individuals, and prospective control data are collected during physical examinations. The present invention recruited hypertensive patients and compared the urine peptide levels of hypertensive patients and normal people with different urine protein excretion levels. Compared with patients with hypertension and proteinuria, the mean urine peptide levels of hypertensive patients without proteinuria were significantly reduced, which is consistent with the reports of patients with hypertension and proteinuria.

[0079] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the present invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for predicting early hypertensive renal damage based on urine peptide determination, characterized in that: Here are the steps: S1: Collect test data of patients with hypertension; S2: Follow up hypertensive patients and confirm their kidney damage; S3: Prepare urine sample and perform test.

2. A method for predicting early hypertensive renal damage based on urine peptide determination according to claim 1, characterized in that: In step S1, data of patients with hypertension are collected and tested, and the steps are as follows: Urine and blood of hypertensive patients were collected and tested, and data of normal population, hypertensive population and hypertension with kidney disease group were collected and screened to form test group data and control group data.

3. A method for predicting early hypertensive renal damage based on urine peptide determination according to claim 2, characterized in that: The data of the test group and the control group included the patient's name, sex, age, HTN duration and hypertension grade, hypertensive fundus lesions, urine microalbumin / creatinine, serum β2-MG, Scr, blood urea nitrogen, homocysteine ​​and cystatin C levels, and estimated glomerular filtration rate calculated using CKD-EPI.

4. A method for predicting early hypertensive renal damage based on urine peptide determination according to claim 1, characterized in that: Hypertensive patients with normoalbuminuria were followed up every 6 months, and blood and first morning urine samples were collected, centrifuged, aliquoted, and frozen.

5. A method for predicting early hypertensive renal damage based on urine peptide determination according to claim 4, characterized in that: To prepare a urine sample and perform the test, follow these steps: A) Bond Elut C18 solid phase extraction cartridges were used to pretreat thawed urine samples at room temperature; B) Using micro-liquid chromatography, 10 μL of urine sample is injected into the mass spectrometer C) quantitative analysis of peak area; D) The abundance values ​​of identified peptides in each sample were combined and quantified using urine creatinine levels; E) Comparisons between different groups.

6. An application of urine peptide determination in predicting early hypertensive renal damage, applying urine peptide determination in kidney testing.

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